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  1. Article ; Online: Elucidating the RNA-Protein Interactomes of Target RNAs in Tissue.

    Whitworth, Isabella T / Henke, Katherine B / Yang, Bing / Scalf, Mark / Frey, Brian L / Jarrard, David F / Smith, Lloyd M

    Analytical chemistry

    2023  Volume 95, Issue 18, Page(s) 7087–7092

    Abstract: RNA-protein interactions are key to many aspects of cellular homeostasis and their identification is important to understanding cellular function. Multiple strategies have been developed for the RNA-centric characterization of RNA-protein complexes. ... ...

    Abstract RNA-protein interactions are key to many aspects of cellular homeostasis and their identification is important to understanding cellular function. Multiple strategies have been developed for the RNA-centric characterization of RNA-protein complexes. However, these studies have all been done in immortalized cell lines that do not capture the complexity of heterogeneous tissue samples. Here, we develop hybridization purification of RNA-protein complexes followed by mass spectrometry (HyPR-MS) for use in tissue samples. We isolated both polyadenylated RNA and the specific long noncoding RNA MALAT1 and characterized their protein interactomes. These results demonstrate the feasibility of HyPR-MS in tissue for the multiplexed characterization of specific RNA-protein complexes.
    MeSH term(s) RNA, Long Noncoding/genetics ; Cell Line ; RNA, Messenger
    Chemical Substances RNA, Long Noncoding ; RNA, Messenger
    Language English
    Publishing date 2023-04-24
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural
    ZDB-ID 1508-8
    ISSN 1520-6882 ; 0003-2700
    ISSN (online) 1520-6882
    ISSN 0003-2700
    DOI 10.1021/acs.analchem.2c05635
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Elucidating the RNA–Protein Interactomes of Target RNAs in Tissue

    Whitworth, Isabella T. / Henke, Katherine B. / Yang, Bing / Scalf, Mark / Frey, Brian L. / Jarrard, David F. / Smith, Lloyd M.

    Analytical Chemistry. 2023 Apr. 24, v. 95, no. 18 p.7087-7092

    2023  

    Abstract: RNA–protein interactions are key to many aspects of cellular homeostasis and their identification is important to understanding cellular function. Multiple strategies have been developed for the RNA-centric characterization of RNA–protein complexes. ... ...

    Abstract RNA–protein interactions are key to many aspects of cellular homeostasis and their identification is important to understanding cellular function. Multiple strategies have been developed for the RNA-centric characterization of RNA–protein complexes. However, these studies have all been done in immortalized cell lines that do not capture the complexity of heterogeneous tissue samples. Here, we develop hybridization purification of RNA–protein complexes followed by mass spectrometry (HyPR-MS) for use in tissue samples. We isolated both polyadenylated RNA and the specific long noncoding RNA MALAT1 and characterized their protein interactomes. These results demonstrate the feasibility of HyPR-MS in tissue for the multiplexed characterization of specific RNA–protein complexes.
    Keywords analytical chemistry ; homeostasis ; hybridization ; mass spectrometry ; messenger RNA ; non-coding RNA ; protein-protein interactions
    Language English
    Dates of publication 2023-0424
    Size p. 7087-7092.
    Publishing place American Chemical Society
    Document type Article ; Online
    ZDB-ID 1508-8
    ISSN 1520-6882 ; 0003-2700
    ISSN (online) 1520-6882
    ISSN 0003-2700
    DOI 10.1021/acs.analchem.2c05635
    Database NAL-Catalogue (AGRICOLA)

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  3. Article: Defining distinct RNA-protein interactomes of SARS-CoV-2 genomic and subgenomic RNAs.

    Whitworth, Isabella T / Knoener, Rachel A / Puray-Chavez, Maritza / Halfmann, Peter / Romero, Sofia / Baddouh, M'bark / Scalf, Mark / Kawaoka, Yoshihiro / Kutluay, Sebla B / Smith, Lloyd M / Sherer, Nathan M

    bioRxiv : the preprint server for biology

    2023  

    Abstract: Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral defense mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different ... ...

    Abstract Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral defense mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA at either of two time points. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. The interactomes indicated viral associations with cell response pathways including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We validated the significance of five protein interactors predicted to exhibit antiviral activity (APOBEC3F, TRIM71, PPP1CC, LIN28B, and MSI2) using siRNA knockdowns, with each knockdown yielding increases in viral production. This study describes new technology for studying SARS-CoV-2 and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.
    Language English
    Publishing date 2023-05-16
    Publishing country United States
    Document type Preprint
    DOI 10.1101/2023.05.15.540806
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Defining Distinct RNA-Protein Interactomes of SARS-CoV-2 Genomic and Subgenomic RNAs.

    Whitworth, Isabella T / Knoener, Rachel A / Puray-Chavez, Maritza / Halfmann, Peter / Romero, Sofia / Baddouh, M'bark / Scalf, Mark / Kawaoka, Yoshihiro / Kutluay, Sebla B / Smith, Lloyd M / Sherer, Nathan M

    Journal of proteome research

    2023  Volume 23, Issue 1, Page(s) 149–160

    Abstract: Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of ... ...

    Abstract Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. Individual interactomes indicated viral associations with cell response pathways, including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We tested the significance of three protein interactors in these pathways (APOBEC3F, PPP1CC, and MSI2) using siRNA knockdowns, with several knockdowns affecting viral gene expression, most consistently PPP1CC. This study describes a new technology for high-resolution studies of SARS-CoV-2 RNA regulation and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.
    MeSH term(s) Humans ; SARS-CoV-2/genetics ; SARS-CoV-2/metabolism ; Subgenomic RNA ; RNA, Viral/genetics ; RNA, Viral/metabolism ; COVID-19/genetics ; Virus Replication/genetics ; Genomics ; RNA-Binding Proteins/genetics
    Chemical Substances Subgenomic RNA ; RNA, Viral ; MSI2 protein, human ; RNA-Binding Proteins
    Language English
    Publishing date 2023-12-03
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
    ZDB-ID 2078618-9
    ISSN 1535-3907 ; 1535-3893
    ISSN (online) 1535-3907
    ISSN 1535-3893
    DOI 10.1021/acs.jproteome.3c00506
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: Defining distinct RNA-protein interactomes of SARS-CoV-2 genomic and subgenomic RNAs

    Whitworth, Isabella T. / Knoener, Rachel A. / Puray-Chavez, Maritza / Halfmann, Peter / Romero, Sofia / Baddouh, M’bark / Scalf, Mark / Kawaoka, Yoshihiro / Kutluay, Sebla B. / Smith, Lloyd M. / Sherer, Nathan M.

    bioRxiv

    Abstract: Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral defense mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different ... ...

    Abstract Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral defense mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA at either of two time points. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. The interactomes indicated viral associations with cell response pathways including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We validated the significance of five protein interactors predicted to exhibit antiviral activity (APOBEC3F, TRIM71, PPP1CC, LIN28B, and MSI2) using siRNA knockdowns, with each knockdown yielding increases in viral production. This study describes new technology for studying SARS-CoV-2 and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.
    Keywords covid19
    Language English
    Publishing date 2023-05-16
    Publisher Cold Spring Harbor Laboratory
    Document type Article ; Online
    DOI 10.1101/2023.05.15.540806
    Database COVID19

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  6. Article ; Online: Defining distinct RNA-protein interactomes of SARS-CoV-2 genomic and subgenomic RNAs

    Whitworth, Isabella T / Knoener, Rachel / Puray Chavez, Maritza / Halfmann, Peter / Romero, Sofia / Baddouh, M'bark / Scalf, Mark / Kawaoka, Yoshihiro / Kutluay, Sebla B / Smith, Lloyd M / Sherer, Nathan M

    bioRxiv

    Abstract: Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral defense mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different ... ...

    Abstract Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral defense mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA at either of two time points. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. The interactomes indicated viral associations with cell response pathways including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We validated the significance of five protein interactors predicted to exhibit antiviral activity (APOBEC3F, TRIM71, PPP1CC, LIN28B, and MSI2) using siRNA knockdowns, with each knockdown yielding increases in viral production. This study describes new technology for studying SARS-CoV-2 and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.
    Keywords covid19
    Language English
    Publishing date 2023-05-16
    Publisher Cold Spring Harbor Laboratory
    Document type Article ; Online
    DOI 10.1101/2023.05.15.540806
    Database COVID19

    Full text online

    Kategorien

    Inter-library loan at ZB MED

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